bioRxiv Science⌕ Search

Biology subjects

van der Laan, M.

Publications and source records attributed to van der Laan, M..

11 recordsLinked to original sources

Exploring the activation mechanism of the Mic60 subcomplex governing mitochondrial crista junctions

Crista junctions (CJs) are narrow infoldings of the inner mitochondrial membrane, serving as entry points into the cristae microenvironment. The mitochondrial contact site and cristae organizing system (MICOS) complex is a highly conserved protein machinery controlling CJ biogenesis and architecture, but its assembly mechanism driving CJ formation has remained unclear. By determining a crystal structure of a Chaetomium thermophilum Mic60-Mic19 complex and combining it with modelling and mutagenesis studies, we identify an extended helical region (EHR) of the Mic60 mitofilin domain and a further Mic19 helix as crucial elements of complex formation. In vitro cross-linking data and hydrogen-deuterium exchange mass spectrometry resolve domain assembly and conformational dynamics of the Mic60 Mic19 subcomplex and point to the EHR as a molecular switch controlling Mic60 transition from a closed dimer to a Mic19-bound hetero-octamer. Our data provide a structural model for Mic60 activation and therefore MICOS assembly as a prerequisite for crista junction formation.

molecular biology↗

Super-resolution complexome of human mitochondria elucidates translocase, morphology and OXPHOS networks

Mitochondria function as cellular powerhouses and central hubs in metabolism, redox and stress reactions, signaling and apoptosis1-5. Defects of mitochondria lead to numerous human diseases1,6-8. The integration of mitochondrial proteins into complexes and networks is crucial for their function. Whereas the composition of the human mitochondrial proteome has been studied8,9, only limited information is available on the organization of the proteome into protein complexes and assemblies. Here we present a systematic mapping of the human mitochondrial complexome from HEK293T cells at super-resolution, resolving more than 7,000 abundance profile peaks of mitochondrial proteins. Proteins functioning in signaling, cell stress, protein biogenesis, turnover and membrane dynamics display particularly high complexities. High resolution and precise quantification enable discrimination between canonical constituents and non-stoichiometric regulatory interactors of the ATP synthase, major metabolite channels and import translocases. The complexome reveals membrane-spanning networks of protein insertase and morphology machinery, and co-assembly of protein import and export components at the major respiratory supercomplex, unraveling a multifunctional organization of mitochondrial machineries. This complexome represents a fully interactive resource for the systematic analysis of human mitochondrial machineries and interaction networks.

biochemistry↗

The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

Over the past decades, chikungunya virus (CHIKV), a re-emerging arthropod-borne alphavirus, has caused outbreaks in many (sub)tropical regions, but also in more temperate regions of the world, including Europe. CHIKV poses a significant health burden due to high infection rates during epidemics, symptom progression into chronic arthritic manifestations, and the lack of specific antiviral treatments. Multiple studies have shown that several viruses rely on host molecular chaperones, particularly the central Heat shock protein 70 (Hsp70), for replication. Hsp70s are guided by co-chaperones that drive their functionality and specificity. Here, we used chemical inhibitors of Hsp70-co-chaperone interactions to study the role of this molecular chaperone machinery in CHIKV replication. Our findings revealed that Hsp70 inhibition significantly reduces the CHIKV infectious particle production without affecting host-cell viability. Inhibition of the Hsp70-co-chaperone interaction primarily impedes the post-RNA replication stages of the CHIKV infectious cycle, affecting viral protein expression and reducing both the number and infectivity of released virions. Moreover, Hsp70 inhibition displayed antiviral activity in skin explants. Together, these results suggest that targeting the Hsp70 network could be a viable antiviral strategy against CHIKV infections. Author summaryAs obligatory intracellular parasites, viruses rely entirely on the machinery of the host cell to produce new viral particles. In our study, we investigated whether a specific group of host proteins, known as molecular chaperones, is important for the replication of chikungunya virus (CHIKV), a reemerging mosquito-borne virus that can cause long-lasting joint pains and lacks specific antiviral treatments. We focused on one key family of molecular chaperones, called Heat shock protein 70s (Hsp70s), which supports protein folding and quality control in cells. We used chemical compounds to block Hsp70 function and observed that CHIKV replication was strongly reduced, while host cells remained healthy. We found that Hsp70 is especially important in the later stages of the virus life cycle, where it helps produce viral proteins and new infectious virus particles. When Hsp70 was blocked, fewer and less infectious virus particles were produced. We also showed that this effect holds true in biopsies of mouse skin tissue, which mimics the initial site of infection. These findings identify Hsp70 as an important host factor for CHIKV that may serve as a potential target for new antiviral therapies.

microbiology↗

Structural studies suggest CCDC127 as a novel membrane contact site protein in the mitochondrial intermembrane space

Mitochondria feature a sophisticated membrane architecture, with a planar mitochondrial outer membrane (MOM) and a folded inner membrane (MIM). Due to the remarkable adaptability of mitochondria, a proteinaceous network in the intermembrane space (IMS) was proposed to confer both stability and flexibility. However, components of such scaffolds, tentatively termed the mitoskeleton, have remained largely elusive. The mitochondrial contact site and organizing system (MICOS), a central organizer of mitochondrial membrane architecture, was suggested to participate in mitoskeleton formation. Here, we structurally characterize the coiled-coil domain-containing 127 (CCDC127) protein, a putative interactor of MICOS. We show that CCDC127s amino-terminal transmembrane region is anchored in the MOM and the bulk soluble part exposed to the IMS. A crystal structure of CCDC127s central coiled-coil displays a parallel dimer which further oligomerizes into tetramers. We demonstrate that the carboxy-terminal helical bundle (CHB) domain dimerizes to create a peripheral membrane-binding site. Supported by electron microscopy data, we propose a structural model of CCDC127 as intramitochondrial membrane contact site protein mediating the structural organization of the IMS as part of the mitoskeleton.

molecular biology↗

Topology control by a conserved cysteine pair in the OMM-protein CCDC127 enables MICOS interaction

Mitochondrial disulfide relay substrates beyond the canonical substrates remain incompletely defined. Revisiting the human MIA40 interactome with enhanced depth, we identified CCDC127 as a previously unrecognized substrate candidate. CCDC127 contains a single transmembrane segment and a conserved C-terminal helical bundle domain (CHB). Comprehensive proteomic and biochemical analyses revealed that, contrary to earlier reports, CCDC127 adopts an Nout-Cin topology in the outer mitochondrial membrane (OMM) with its CHB residing in the intermembrane space (IMS). CCDC127 undergoes oxidation by the disulfide relay, forming a long-range intramolecular disulfide bond between C174 and C219. Loss of these cysteines disrupts correct OMM insertion, inverts transmembrane topology and triggers proteasome-dependent degradation, establishing the disulfide as a key determinant of CCDC127 maturation. Interactome analyses identified MICOS components--particularly the MIC60/MIC19 module--as major partner proteins required for the stability of large oligomeric CCDC127 complexes. CCDC127 deficiency impaired cellular proliferation, influenced phospholipid levels, and caused grossly altered cristae morphology. Together, CCDC127 emerges as a MICOS-associated OMM protein essential for mitochondrial membrane organization and lipid homeostasis.

biochemistry↗

A multiplexed approach for genetic screening of human cells by electron microscopy uncovers a critical effector of mitochondrial cristae shape

Mitochondria form complex and diverse membrane-architectures essential for their multiple functions. Whereas critical proteins sculpting mitochondrial membranes have been identified, the molecular basis for many key features remains enigmatic. Exploration of membrane ultrastructure, in general, is limited by the tradeoff between resolution and throughput: electron microscopy (EM) is essential to resolve their ultrastructure but lacks scalability for systematic functional discovery. To overcome this limitation, we developed a high-resolution screening pipeline for EM of multiplexed human cell pools, hMultiCLEM (human Multiplexed Correlative Light and EM). To showcase the power of hMultiCLEM we performed a genetic screen exploring mitochondrial ultrastructure. hMultiCLEM confirmed proposed cristae modulators and uncovered additional ones illuminating the protein networks driving cristae organization. Validation of candidates highlighted an intermembrane space (IMS) protein linked to Menieres disease, which we named MISHA (Mitochondrial-IMS membrane-SHApe-impacting protein). More broadly, hMultiCLEM transforms the EM field, enabling genetic/chemical screening in basic and medical research.

cell biology↗

Pancreatic islets undergo functional and morphological adaptation during development of Barth Syndrome

Barth syndrome is a multisystem genetic disorder caused by mutation in TAFAZZIN, a gene that encodes a phospholipid:lysophospholipid transacylase important for cardiolipin remodeling. Barth Syndrome patients suffer from a number of symptoms including early heart failure, fatigue, and systemic metabolic alterations, including hypoglycemia. The endocrine pancreas is central to glucose homeostasis, however, the impact of defective cardiolipin remodeling on pancreatic islet function and the consequences for systemic metabolism is unclear. Surprisingly, in a mouse model with global TAFAZZIN knockdown, we observed improved glucose tolerance compared to wildtype littermates. We show that pancreatic islet metabolism and secretory function are robustly maintained through various compensatory mechanisms including increased glucose uptake and increased mitochondrial volume. Transcriptomics analyses revealed increased expression of genes encoding proteins involved in N-acetylglucosamine synthesis and protein O-linked N-acetylglucosaminylation. These pathways might provide a molecular mechanism for coupling metabolic changes to mitochondrial volume regulation.

physiology↗

Coordination of cytochrome bc1 complex assembly at MICOS

The boundary and cristae domains of the mitochondrial inner membrane are connected by crista junctions. Most cristae membrane proteins are nuclear-encoded and inserted by the mitochondrial protein import machinery into the inner boundary membrane. Thus, they must overcome the diffusion barrier imposed by crista junctions to reach their final location. Here, we show that respiratory chain complexes and assembly intermediates are physically connected to the mitochondrial contact site and cristae organizing system (MICOS) that is essential for formation and stability of crista junctions. We identify the inner membrane protein Mar26 (Fmp10) as determinant in the biogenesis of the cytochrome bc1 complex (complex III). Mar26 couples a Rieske Fe/S protein-containing assembly intermediate to MICOS. Our data indicate that Mar26 maintains an assembly-competent Rip1 pool at crista junctions where complex III maturation likely occurs. MICOS facilitates efficient Rip1 assembly by recruitment of complex III assembly intermediates to crista junctions. We propose that MICOS, via interaction with assembly factors such as Mar26, directly contributes to the spatial and temporal coordination of respiratory chain biogenesis.

biochemistry↗

Deep surveys of transcriptional modules with Massive Associative Kbiclustering (MAK)

Biclustering can reveal functional patterns in common biological data such as gene expression. Biclusters are ordered submatrices of a larger matrix that represent coherent data patterns. A critical requirement for biclusters is high coherence across a subset of columns, where coherence is defined as a fit to a mathematical model of similarity or correlation. Biclustering, though powerful, is NP-hard, and existing biclustering methods implement a wide variety of approximations to achieve tractable solutions for real world datasets. High bicluster coherence becomes more computationally expensive to achieve with high dimensional data, due to the search space size and because the number, size, and overlap of biclusters tends to increase. This complicates an already difficult problem and leads existing methods to find smaller, less coherent biclusters. Our unsupervised Massive Associative K-biclustering (MAK) approach corrects this size bias while preserving high bicluster coherence both on simulated datasets with known ground truth and on real world data without, where we apply a new measure to evaluate biclustering. Moreover, MAK jointly maximizes bicluster coherence with biological enrichment and finds the most enriched biological functions. Another long-standing problem with these methods is the overwhelming data signal related to ribosomal functions and protein production, which can drown out signals for less common but therefore more interesting functions. MAK reports the second-most enriched non-protein production functions, with higher bicluster coherence and arrayed across a large number of biclusters, demonstrating its ability to alleviate this biological bias and thus reflect the mediation of multiple biological processes rather than recruitment of processes to a small number of major cell activities. Finally, compared to the union of results from 11 top biclustering methods, MAK finds 21 novel S. cerevisiae biclusters. MAK can generate high quality biclusters in large biological datasets, including simultaneous integration of up to four distinct biological data types. Author summaryBiclustering can reveal functional patterns in common biological data such as gene expression. A critical requirement for biclusters is high coherence across a subset of columns, where coherence is defined as a fit to a mathematical model of similarity or correlation. Biclustering, though powerful, is NP-hard, and existing biclustering methods implement a wide variety of approximations to achieve tractable solutions for real world datasets. This complicates an already difficult problem and leads existing biclustering methods to find smaller and less coherent biclusters. Using the MAK methodology we can correct the bicluster size bias while preserving high bicluster coherence on simulated datasets with known ground truth as well as real world datasets, where we apply a new data driven bicluster set score. MAK jointly maximizes bicluster coherence with biological enrichment and finds more enriched biological functions, including other than protein production. These functions are arrayed across a large number of MAK biclusters, demonstrating ability to alleviate this biological bias and reflect the mediation of multiple biological processes rather than recruitment of processes to a small number of major cell activities. MAK can generate high quality biclusters in large biological datasets, including simultaneous integration of up to four distinct biological data types.

bioinformatics↗

TLR2 axis on peripheral blood mononuclear cells regulates inflammatory responses to circulating, non-infectious immature dengue virus particles

Severe dengue virus (DENV) infection is characterized by exacerbated inflammatory responses that lead to endothelial dysfunction and plasma leakage. We have recently demonstrated that Toll-like receptor 2 (TLR2) on blood monocytes senses DENV infection leading to endothelial activation. Here, we report that non-infectious immature DENV particles, which are released in large numbers by DENV-infected cells, drive endothelial activation via the TLR2 axis. We show that fully immature DENV particles induce a rapid, within 6 hours post-infection, inflammatory response in PBMCs. Furthermore, pharmacological blocking of TLR2/TLR6/CD14 and/or NF-kB prior to exposure of PBMCs to immature DENV reduces the initial production of inter alia TNF- and IL-1{beta} by monocytes and prevents endothelial activation. However, prolonged TLR2 block induces TNF- production and leads to exacerbated endothelial activation, indicating that TLR2-mediated responses play an important role not only in the initiation but also the resolution of inflammation. Altogether, these data indicate that the maturation status of the virus has the potential to influence the kinetics and extent of inflammatory responses during DENV infection. Author SummarySevere dengue virus (DENV) infection is characterized by endothelial dysfunction leading to vascular permeability and plasma leakage. This is thought to be mediated by the exacerbated production of inflammatory mediators from cells of the innate immune system. We have previously demonstrated that Toll-like receptor 2 (TLR2), a pattern recognition receptor present on the surface of human monocytes, can sense DENV infection leading to the activation of the endothelium. Importantly, a large proportion of DENV particles is immature and are not readily infectious. Here we aimed to elucidate if and how these non-infectious immature DENV particles contribute to systemic inflammation. We evaluated if monocytes sense immature DENV and found that sensing of immature DENV induced early inflammatory responses in PBMCs. Pharmacological inhibition of TLR2/TLR6/CD14 and/or NF-{kappa}B prior to exposure to immature DENV demonstrates that this is the pathway leading to the early production inflammatory mediators and endothelial activation. However, prolonged inhibition of TLR2 induced the production of TNF- and the subsequent activation of the endothelium, suggesting that TLR2-mediated responses play an important tole during both, initiation and resolution of inflammation. We propose that the maturation status of DENV in the human host can influence the extent and kinetics of the inflammatory responses during DENV infection.

immunology↗

Structural insights into crista junction formation by the Mic60-Mic19 complex

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil domain of the mitochondrial contact and cristae organizing system (MICOS) subunit Mic60 forms an elongated, bow tie-shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with curved CJ necks. Our study suggests that the Mic60-Mic19 subcomplex transverses CJs as a molecular strut, thereby controlling CJ architecture and function.

cell biology↗